ABSTRACT Traumatic brain injury (TBI) is an important public health problem. Currently, the ability to objectively assess TBI is a critical research gap. CT and conventional structural MRI have proven to be highly effective in identifying macroscopic lesions. However, these standard imaging techniques have clear limitations in assessing important microscopic lesions and neurologic pathology. The clinical diagnosis of TBI via imaging, particularly mild TBI, remains controversial, because the brain often appears quite normal on conventional CT and MRI. Therefore, new sensitive surrogate biomarkers for TBI are greatly needed in routine clinical practice. Amide proton transfer (APT) imaging is an important molecular MRI technique that can generate contrast based on tissue pH or concentrations of endogenous mobile proteins and peptides. In our preliminary study, we have applied the APTw-MRI approach to a rat TBI model, induced by controlled cortical impact (CCI). Our preliminary results have demonstrated unique APTw-MRI signal characteristics at different time points after injury that are associated with ischemia (at a few hours) and neuroinflammation (at 2-3 days). Notably, APT imaging revealed an acidosis-based ischemic penumbra around the impacted area at a few hours post-injury. These initial results are very promising. However, further development and radiographic-histopathologic validation with different TBI models and at other research sites is crucial for translating this innovative technology and these important results to the clinic. The overall goal of this application is to demonstrate the feasibility, potential, and reproducibility of protein-based APT-MRI signals as functional markers for TBI using animal models of mild, moderate, or severe TBI. We hypothesize that molecular imaging using APT-MRI can sensitively and non-invasively visualize ischemic damage, inflammatory responses, and several other key pathological processes in TBI, thus improving the capability of MRI to objectively assess TBI. Our three specific aims are: (i) to assess APT-MRI spatio-temporal evolution characteristics of TBI and the underlying pathological mechanisms in rat CCI models; (ii) to assess whether APT-MRI predicts therapeutic outcomes in rat CCI models; and (iii) to validate the sensitivity and reliability of APT-MRI in assessing TBI at external sites. Molecular imaging of TBI using APT-MRI opens up a new research area of APT imaging that could address many unmet clinical needs. If our aims in this preclinical study are achieved, the results will provide the solid foundation required to translate this important MRI technology to clinical studies in patients with TBI.